Table S1 Sequences of the oligonucleotides used for construction of the IbAGP1 promoter deletion plasmids
Oligo names / Primer sequences / Insertion fragment sizeIbAGP1-F1 / 5’-GCAAGCTTACTGATACTTTTGGTGACTGC-3’ / 1989bp
IbAGP1-F2 / 5’-GCAAGCTTCTTTGGTTTCTTTATTTATG-3’ / 1673bp
IbAGP1-F3 / 5’-GCAAGCTTCGAGAATTACTACCATTCTC-3’ / 1373bp
IbAGP1-F4 / 5’-GCAAGCTTGGTCTGCATTTCACGTGGGG-3’ / 1128bp
IbAGP1-F5 / 5’-GCAAGCTTCTGTATCATGTAAATTTTAT-3’ / 791bp
IbAGP1-F6 / 5’-GCAAGCTTGATATACTCCGTATAAGTAA-3’ / 361bp
IbAGP1-R7 / 5’-GGATCCCATTTTTAAGCCGCGCTACC-3’
*The HindIII and BamHIrestriction enzyme site is underlined.
Table S2 Sequences of the oligonucleotides used for construction of the artificial promoters
Artificial promoter names / Oligo names / Primer sequences / Artificial fragment size4XSURE-Like / ZY277-F / aattcAATAAATAAAcaatAATAAAAAAcaatAATAAAAcaatAATAAAAG / 57bp
ZY277-R / GATCcTTTTATTattgTTTTATTattgTTTTTTATTattgTTTATTTATTg
4XW box / ZY279-F / aattcTGACTcaatTGACTcaatTGACTcaatTGACTG / 44bp
ZY279-R / GATCcAGTCAattgAGTCAattgAGTCAattgAGTCAg
35S m / ZY275-F / aattcAGGATCCgcaagacccttcctctatataaggaagttcatttcatttggagaggT / 46bp
ZY275-R / CTAGAcctctccaaatgaaatgaacttccttatatagaggaagggtcttgcGGATCCTg
*TheEcoRI, BamHI andXbaIrestriction enzyme site is underlined.
Table S3 Regulatory elements and transcription factor binding sites in the of the IbAGP1 promoter
Motifs / Organism / Sequence / position / DescriptionABRE / Arabidopsis thaliana / TACGTG / -1386 / cis-acting element involved in the abscisic acid responsiveness
ABRE / Arabidopsis thaliana / CACGTG / -1041 / cis-acting element involved in the abscisic acid responsiveness
AuxRR-core / Nicotiana tabacum / GGTCCAT / -998 / cis-acting regulatory element involved in auxin responsiveness
Box 4 / Petroselinum crispum / ATTAAT / -334 / part of a conserved DNA module involved in light responsiveness
Box I / Pisum sativum / TTTCAAA / -1333 / light responsive element
CAT-box / Arabidopsis thaliana / GCCACT / -1007 / cis-acting regulatory element related to meristem expression
CAAT-box / Glycine max / CAATT / -1624, -1410 / common cis-acting element in promoter and enhancer regions
CAAT-box / Hordeum vulgare / CAAT / -1740, -1643, -1532, -1410, -1318, -888, -829, -773, -371 / common cis-acting element in promoter and enhancer regions
CATT-motif / Zea mays / GCATTC / -1099, -733 / part of a light responsive element
CGTCA-motif / Hordeum vulgare / CGTCA / -1766, -1012 / cis-acting regulatory element involved in the MeJA-responsiveness
G-Box / Pisum sativum / CACGTG / -1041 / cis-acting regulatory element involved in light responsiveness
G-box / Daucus carota / TACGTG / -1386 / cis-acting regulatory element involved in light responsiveness
GAG-motif / Spinacia oleracea / AGAGATG / -1524 / part of a light responsive element
Skn-1_motif / Oryza sativa / GTCAT / -1765, -1660, -1648 / cis-acting regulatory element required for endosperm expression
GACG-motif / Hordeum vulgare / TGACG / -1178 / cis-acting regulatory element involved in the MeJA-responsiveness
ROOT-motif / Agrobacterium rhizogenes / ATATT / -935, -400, -245 / Motif found both in promoters of rolD and domain A of the 35S promoter
Sp1 / Zea mays / CCACCC / -173 / light responsive element
SURE-like / Solanum tuberosum / AATAAAA / -609, -189 / sucrose responsive element
SURE-like / Solanum tuberosum / AATAAAAAA / -681 / sucrose responsive element
SURE-like / Solanum tuberosum / AATAAATAAA / -1273, -1239 / sucrose responsive element
SRE-box / Arabidopsis thaliana / TTATC / -726, -700, -356 / Sugar-repressive relative element
TCT-motif / Arabidopsis thaliana / TCTTAC / -1687, -349 / part of a light responsive element
W-box / Arabidopsis thaliana / TGACT / -1985, -1434, -750, -578 / Site of binding of WRKYs transcription factors
*Identification of putative regulatory elements was achieved by in silico analysis of the full IbAGP1 promoter sequences using PlantCARE and PLACE databases.
TableS4 Activity of GUS (pmol MU•min-1•mg protein-1)
Line / Tissues / Uninducible / Inducible / Fold change of activity of GUS*pBI121 / L / 339.219±34.540 / 342.871±27.879 / 1.011
S / 352.029±19.414 / 376.200±21.838 / 1.069
R / 264.383±15.592 / 264.536±18.070 / 1.000
WT / L / 7.218±1.548 / 7.133±1.952 / 0.988
S / 5.083±1.123 / 4.076±0.611 / 0.801
R / 7.203±1.627 / 7.766±1.982 / 1.078
AGP1-Ⅰ / L / 106.484±21.967 / 746.308±54.441 / 7.009
S / 97.361±21.920 / 818.482±48.296 / 8.407
R / 115.752±20.863 / 298.571±51.240 / 2.579
AGP1-Ⅱ / L / 86.139±11.521 / 313.972±42.337 / 3.645
S / 77.610±13.274 / 316.878±36.420 / 4.083
R / 97.844±9.553 / 188.698±26.632 / 1.929
AGP1-Ⅲ / L / 59.513±16.200 / 210.295±29.608 / 3.534
S / 57.566±4.606 / 222.825±37.356 / 4.083
R / 68.295±17.618 / 135.770±30.759 / 1.988
AGP1-Ⅳ / L / 49.494±13.050 / 114.110±36.438 / 2.306
S / 51.655±14.853 / 125.590±19.792 / 2.431
R / 53.896±14.313 / 129.312±11.644 / 2.400
AGP1-Ⅴ / L / 22.668±8.214 / 84.840±13.414 / 3.743
S / 18.330±1.817 / 96.351±13.071 / 5.256
R / 27.512±6.049 / 109.630±16.142 / 3.958
AGP1-Ⅵ / L / 9.580±2.400 / 31.565±2.220 / 3.295
S / 6.140±0.317 / 25.122±5.079 / 4.092
R / 10.479±3.666 / 86.160±17.689 / 8.222
*Fold change of activity of GUS (c=b/a).
L representatives leaves, S representatives stems, R representatives roots.
Different tissues of at least threeindividual plants were measured. Data are analyzed by Student’s t-test, means±SD from three separate experiments. P< 0.05.
Fig. S1 PCR screening for transgenic tobacco plants. PCR amplification of the GUS transgene was performed in transgenic plants carrying different AGP1::GUS constructs. The amplicon of 999 bp was found in most transgenic lines. Plasmid , a positive control using plasmid DNA as template; WT, wild type (a negative control); lanes 1-8, eight independent transgenic lines for each construct. Results for all seven constructs corresponding to pBI121, IbAGP1-I, IbAGP1-II, IbAGP1-III, IbAGP1-IV, IbAGP1-V, and IbAGP1-VI are shown in separate panels.